Most excitatory synaptic transmission in the mammalian central nervous system is mediated by the neurotransmitter glutamate. Glutamatergic signaling is critical to fast cell-to-cell transmission, normal brain development, and learning and memory. Dysfunctional glutamatergic signaling is implicated in numerous acute and chronic neurological diseases as well as many psychiatric disorders. NMDA receptors (NMDARs) are glutamate activated ion channels (iGluRs) that are integral to this fast signaling. A key functional feature of NMDARs is gating - the process of ligand binding/unbinding resulting in pore opening/closing. Gating involves ligand induced conformational changes in the ligand binding domain that are transferred to the pore forming transmembrane domain, increasing the likelihood of channel opening. This gating process is a promising target for pharmacological intervention. I will address the novel hypothesis that electrostatic interactions in the linkers connecting the ligand binding domain to the transmembrane domain (specifically, M3-S2 and S2-M4) strongly influence the energetics of gating in NMDARs. In GluN2A subunits, the M3-S2 and S2-M4 linkers have numerous charged residues and I have preliminary data suggesting that these linkers are proximal and that gating kinetics are significantly altered if charges are mutated. Aim 1 will focus on the detailed mechanisms of how electrostatic interactions between charged residues in the M3-S2 and S2- M4 linkers affect gating. I will use single channel analysis, immunoblots, and substituted cysteines to determine how these charged residues interact to modulate gating energetics. In Aim 2, I will explore how electrostatic interactions in the linkers may contribute to subunit- specific gating mechanisms. The GluN2 and GluN3 subunits confer distinct gating properties onto NMDARs and define how NMDARs function at native synapses. I hypothesize that differences in intrasubunit linker electrostatic interactions are part of the underlying mechanism of subunit-specific gating. I will take advantage of insights gained from Aim 1 and single-channel recordings to test how electrostatic interactions in the linkers might be subunit-specific. Overall, the knowledge gained from these studies will provide significant insight into NMDAR gating and open avenues for potential sites of pharmacological intervention that are both subtype and subunit specific.